A basic principle of flow cytometry is the passage of cells in a fluid-stream through a focused laser-beam so that the cells can be detected, identified, counted, and sorted. Cell components are fluorescently labelled and then excited by the laser-beam to emit light at varying wavelengths. The fluorescence can then be measured to determine the amount and type of cells present in a sample. Up to thousands of particles per second can be analyzed as they pass through the fluid-stream.
Several detectors are carefully placed around the fluid-stream at the point where the fluid passes through the focused beam. The suspended particles or cells, which may range in size from 0.2 micrometers (μm) to 150 μm, pass through the focused beam and scatter the radiation. The fluorescently labelled cell components are also excited by the focused laser-beam and emit light (fluorescence) at a longer wavelength than that of the laser-beam. The fluorescence is then detected by the detectors. The detectors measure a combination of scattered and fluorescent light. Measurement data is then analyzed, using special software, by a computer that is attached to the flow cytometer.
It is generally accepted that the above described flow cytometry process is more flexible and more accurate the more light-wavelengths that are included in the laser-beam. In practice, this is accomplished by combining component beams from different lasers along a common path to provide a combined beam that is focused into the fluid-stream. Diode-laser modules are typically used for providing the component beams. Commercially available diode-laser modules can provide laser radiation at selected fundamental wavelengths in a range from the near ultraviolet (UV) the near infrared (NIR).
An increasing number and range of wavelengths presents significant problems in the design and construction of an optical objective for focusing the combined laser-beam into the fluid-stream. It is generally accepted that for focusing two significantly different wavelengths at a common location (focal plane) a focusing objective must include at least two lens elements having different, for example high and low, spectral dispersion. An objective arranged to focus two different wavelengths (red and blue) in a common focal plane is generally referred to as an achromatic objective.
If three significantly different wavelengths, for example, red, green, and blue wavelengths, are to be focused at a common location, a focusing objective must include at least three lens elements having different spectral dispersion. An objective arranged to focus three significantly different wavelengths (red, green, and blue) in a common focal plane is generally referred to as an apochromatic objective.
In either achromatic or apochromatic objectives individual (singlet) lens elements of different spectral dispersion may need to be “cemented” together in a form referred to by practitioners of the lens design art as “doublets” or “triplets”. This could provide a problem in including UV wavelengths in a flow cytometer, as optical cements (adhesives) may be degraded by the UV radiation
Based on conventional optical design wisdom, it can be expected that as more laser-radiation wavelengths, for example four or more, are included in a flow cytometer, the more complex and expensive will be the objective required to focus the wavelengths into the fluid-stream. This could result in the cost and complexity of a focusing objective determining a practical upper limit to how many laser-radiation wavelengths could be used in a flow-cytometer.
There is need for a simple focusing objective, capable of focusing four or more laser radiation wavelengths in a common focal plane, but wherein the number of different optical materials (glasses) required is less than the number of different wavelengths to be focused by the objective in the common focal plane. Preferably the focusing objective should not include any cemented doublet or triplet elements.